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Background  Eighteen imported ovale malaria cases imported from Myanmar and various African countries have been reported in Yunnan Province, China from 2013 to 2018. All of them have been confirmed by morphological examination and 18S small subunit ribosomal RNA gene (18S rRNA) based PCR in YNRL. Nevertheless, the subtypes of  Plasmodium ovale  could not be identified based on 18S rRNA gene test, thus posing challenges on its accurate diagnosis. To help establish a more sensitive and specific method for the detection of  P. ovale  genes, this study performs sequence analysis on  k13 -propeller polymorphisms in  P. ovale.Methods  Dried blood spots (DBS) from ovale malaria cases were collected from January 2013 to December 2018, and the infection sources were confirmed according to epidemiological investigation. DNA was extracted, and the coding region (from 206th aa to 725th aa) in  k13  gene propeller domain was amplified using nested PCR. Subsequently, the amplified products were sequenced and compared with reference sequence to obtain CDS. The haplotypes and mutation loci of the CDS were analysed, and the spatial structure of the amino acid peptide chain of  k13  gene propeller domain was predicted by SWISS-MODEL.    Results  The coding region from 224th aa to 725th aa of  k13  gene from  P. ovale  in 83.3% of collected samples (15/18) were amplified. Three haplotypes were observed in 15 samples, and the values of Ka/Ks, nucleic acid diversity index (π) and expected heterozygosity (He) were 3.784, 0.0095, and 0.4250.  Curtisi  haplotype,  Wallikeri  haplotype, and mutant type accounted for 73.3% (11/15), 20.0% (3/15), and 6.7% (1/15). The predominant haplotypes of  P. ovale curtisi  were determined in all five Myanmar isolates. Of the ten African isolates, six were identified as  P. o. curtisi , three were  P. o. wallikeri  and one was mutant type. Base substitutions between the sequences of  P. o. curtisi  and  P. o. wallikeri  were determined at 38 loci, such as c.711. Moreover, the A >   T   base substitution at c.1428 was a nonsynonymous mutation, resulting in amino acid variation of T476S in the 476th position. Compared with sequence of  P. o. wallikeri , the double nonsynonymous mutations of G >   A   and A >   T   at the sites of c.1186 and c.1428 leads to the variations of D396N and T476S for the 396th and 476th amino acids positions. For  P. o. curtisi  and  P. o. wallikeri , the peptide chains in the coding region from 224th aa to 725th aa of  k13  gene merely formed a monomeric spatial model, whereas the double-variant peptide chains of D396N and T476S formed homodimeric spatial model.    Conclusion  The propeller domain of  k13  gene in the  P. ovale  isolates imported into Yunnan Province from Myanmar and Africa showed high differentiation. The sequences of Myanmar-imported isolates belong to  P. o. curtisi , while the sequences of African isolates showed the sympatric distribution from  P. o. curtisi ,  P. o. wallikeri  and mutant isolates. The CDS with a double base substitution formed a dimeric spatial model to encode the peptide chain, which is completely different from the monomeric spatial structure to encode the peptide chain from  P. o. curtisi  and  P. o. wallikeri .

malaria journal

Polymorphism analysis of propeller domain of k13 gene in Plasmodium ovale curtisi and Plasmodium ovale wallikeri isolates original infection from Myanmar and Africa in Yunnan Province, China